Acute toxicity data of common agricultural insecticides to Japanese wild bees

Although numerous ecotoxicological assessments of European honeybee (Apis mellifera L.) have been performed, Japanese wild bees are less well studied in this regard. To address this knowledge gap, we assessed the mortality and acute toxicity (LD50) of 3 common agricultural insecticides (clothianidin, fipronil, and diazinon) on as many as 6 species of Japanese wild bees (Andrena prostomias Perez, Apis cerana japonica Radoszkowski, Bombus deuteronymus Perez, Bombus honshuensis Tkalcu, Bombus hypocrita Perez, and Eucera spp.; all or any of them). The datasets were obtained via standard acute toxicity testing, with assessment of mortality at 24 and 48 h after exposure to the insecticides. These data provide important information regarding the effects of insecticides on Japanese wild bees and their conservation.


Specifications
Biology Specific subject area Ecotoxicology Type of data

Value of the Data
• Honeybees and other wild bees are among the most effective pollinators, providing essential ecosystem services for wild plant reproduction and agriculture production [ 1 , 2 ]. Although data regarding the exposure of honeybees (especially Apis mellifera ) to insecticides have been accumulating in recent years, that of other Japanese wild bees have been overlooked. Our data provide ecotoxicological data on some of these less-studied species. • Although the Ministry of the Environment (MoE) in Japan requires ecotoxicological data of wild bees for their conservation [3] , few of these data have been collected. Therefore, the acute toxicity data that we have collected for 6 Japanese wild bees likely will be highly beneficial for MoE and conservation ecologists. • We anticipate that our ecotoxicological assessment of 6 Japanese wild bee species will be used for Species Sensitivity Distribution analysis (SSD) [4] and help to refine the ecotoxicological assessment of pesticides on wild bees in Japan.

Objective
Although pollinators like bees provide essential ecosystem service for wild plant reproduction and agricultural production [ 1 , 2 ], ecotoxicological data for conservation of these wild bees are lacking in Japan [3] . Our objective is to obtain acute toxicity data for 6 Japanese wild bee species ( Andrena prostomias, Apis cerana japonica, Bombus deuteronymus, Bombus honshuensis, Bombus hypocrita, and Eucera spp.) after exposure to a maximum of 3 common agricultural insecticides (clothianidin, fipronil, and diazinon) in Japan. Table 1 : Details of sampling sites (coordinates), dates, and exposed insecticides of Andrena prostomias, Apis cerana japonica, Bombus deuteronymus, Bombus honshuensis, Bombus hypocrita, and Eucera spp . Table 2 : Mortality rates of those bee species (some or all of them) at 24 and 48 h after exposure to clothianidin, fipronil, and diazinon. Table 3 : 50% lethal dose (LD 50 ) of each insecticide for each bee species as estimated from the mortality data in Table 2 .

Data Description
The data file containing these tables is available in Mendeley Data [5] .

Sample Collection
From May through September 2019, workers (sterile females) of Bombus deuteronymus, Bombus honshuensis , and Bombus hypocrita and females of Andrena prostomias and Eucera spp. were collected in their natural habitat; collection sites are listed in Table 1 . Workers of Apis cerana japonica were collected from reared colonies in our laboratory; A. cerana japonica brood combs were cut into small pieces and kept in a dark incubator (35 °C) to allow adult worker bees to emerge [6] . Emerged workers were maintained at 25 °C in an incubator until acute toxicity tests were conducted. As mentioned earlier, we conducted acute toxicity tests by using fieldcollected adults (except for A. cerana japonica ). Therefore, differences in the ages (no. of days post-hatching) of bees may have affected their susceptibility to insecticides.

Acute Toxicity Test
We conducted all acute toxicity tests at the same location (NIES). Most (but not all) species were tested on the same day ( Table 2 ). The active ingredients of each insecticide (clothianidin, 99.7%; fipronil, 99.4%; diazinon, 99.4%; FUJIFILM Wako Pure Chemical Corporation) were adjusted to the indicated concentrations ( Table 2 ) by using acetone. Bees were placed individually and housed in plastic cups ( ϕ86 × 40 mm), allowed to acclimate for more than 12 h, and then mildly anesthetized by chilling at 5 °C for 15 min before insecticide application. According to OECD test guidelines [7] , 2 μL of diluted insecticide solution should be applied to the dorsal thorax of a bee. However, two of the bee species ( Andrena prostomias, Eucera spp.) we evaluated were too small and hairy to smoothly apply insecticides onto the dorsal thorax. This situation suggests that the evaporation of the acetone is greater in species with smaller body size, making it difficult to ensure uniform insecticide exposure among species. To overcome this problem, we applied 2 μL of diluted insecticide solution to the abdomen of bees, which was easy to accomplish regardless of the bee species. Additional bees were treated with acetone only, as controls. After application, bees were housed in individual plastic cups that had filter paper at the bottom and a 1.5-mL microtube containing a 50% (w/v) solution of sucrose in water as food. Bees were kept under thermostatic conditions of 22 °C (light:dark, 16:8) for Andrena prostomias and Eucera spp. and of 25 °C (total darkness) for the other 4 bee species. At 24 and 48 h after insecticide application, plastic cups were briefly removed from the incubator to record the number of bees that had died by visual observation, and mortality rates for each time point and insecticide concentration were calculated ( Table 2 ). When the mortality in the control group (0 in Dose column, Table 2 ) exceeded 10%, the experimental results were excluded from the data set (i.e., in the n = 5 control group, if even one bee died, the experimental results were excluded from the data set). Moreover, mortality of the treatment group was corrected by using Abbott's formula [8] , corr ected mort ality = mortality of treatment group − mortality of control group 1 − mortality of control group and any correction that resulted in a negative calculated value was converted to zero mortality [9] . Note that although we collected enough number of bees for our study, we were unable to obtain mortality and toxicity data for all combinations of 3 insecticides x 6 bee species. Because some of them died during transport to our laboratory and some combinations resulted in higher mortality ( > 10%) in the control groups as a result of acute toxicity tests ( Table 2 ). After completion of acute toxicity testing, each tested bee was weighed individually to calculate LD 50 values on a per-weight basis (see Statistical analysis ).